The integrins are a class of membrane-associated molecules which actively participate in cellular adhesion. Integrins are transmembrane heterodimers comprising an xcex1 subunit in noncovalent association with a xcex2 subunit. To date, at least fourteen xcex1 subunits and eight xcex2 subunits have been identified [reviewed in Springer, Nature 346:425-434 (1990)]. The xcex2 subunits are generally capable of association with more than one xcex1 subunit and the heterodimers sharing a common xcex2 subunit have been classified as subfamilies within the integrin population.
One class of human integrins, restricted to expression in white blood cells, is characterized by a common xcex22 subunit. As a result of this cell-specific expression, these integrins are commonly referred to as the leukocyte integrins, Leu-CAMs or leukointegrins. Because of the common xcex22 subunit, an alternative designation of this class is the xcex22 integrins. The xcex22 subunit (CD18) has previously been isolated in association with one of three distinct xcex1 subunits, CD11a, CD11b or CD11c. The isolation of a cDNA encoding human CD18 is described in Kishimoto, et al., Cell 48:681-690 (1987). In official WHO nomenclature, the heterodimeric proteins are referred to as CD11a/CD18, CD11b/CD18, and CD11c/CD18; in common nomenclature they are referred to as LFA-1, Mac-1 or Mol and p150,95 or LeuM5, respectively [Cobbold, et al., in Leukocyte Typing III, McMichael (ed), Oxford Press, p.788 (1987)]. The human xcex22 integrin xcex1 subunits CD11a, CD11b and CD11c have been demonstrated to migrate under reducing condition in electrophoresis with apparent molecular weights of approximately 180 kD, 155 kD and 150 kD, respectively, and DNAs encoding these subunits have been cloned [CD11a, Larson, et al., J. Cell Biol. 108:703-712 (1989); CD11b, Corbi, et al., J.Biol.Chem. 263:12403-12411 (1988) and CD11c, Corbi, et al. EMBO J. 6:4023-4028 (1987)]. Putative homologs of the human xcex22 integrin xcex1 and xcex2 chains, defined by approximate similarity in molecular weight, have been variously identified in other species including monkeys and other primates [Letvin, et al., Blood 61:408-410 (1983)], mice [Sanchez-Madrid, et al., J.Exp.Med. 154:1517 (1981)], and dogs [Moore, et al., Tissue Antigens 36:211-220 (1990)].
The absolute molecular weights of presumed homologs from other species have been shown to vary significantly [see, e.g., Danilenko et al., Tissue Antigens 40:13-21 (1992)], and in the absence of sequence information, a definitive correlation between human integrin subunits and those identified in other species has not been possible. Moreover, variation in the number of members in a protein family has been observed between different species. Consider, for example, that more IgA isotypes have been isolated in rabbits than in humans [Burnett, et al., EMBO J. 8:4041-4047 (1989) and Schneiderman, et al., Proc.Natl.Acad.Sci.(USA) 86:7561-7565 (1989)]. Similarly, in humans, at least six variants of the metallothionine protein have been previously identified [Karin and Richards, Nature 299:797-802 (1982) and Varshney, et al., Mol.Cell.Biol. 6:26-37, (1986)], whereas in the mouse, only two such variants are in evidence [Searle, et al., Mol.Cell.Biol. 4:1221-1230 (1984)]. Therefore, existence of multiple members of a protein family in one species does not necessarily imply that corresponding family members exist in another species.
In the specific context of xcex22 integrins, in dogs it has been observed that the presumed canine xcex22 counterpart to the human CD18 is capable of dimer formation with as many as four potentially distinct xcex1 subunits [Danilenko, et al., supra]. Antibodies generated by immunizing mice with canine splenocytes resulted in monoclonal antibodies which immunoprecipitated proteins tentatively designated as canine homologs to human CD18, CD11a, CD11b and CD11c based mainly on similar, but not identical, molecular weights. Another anti-canine splenocyte antibody, Ca11.8H2, recognized and immunoprecipitated a fourth xcex1-like canine subunit also capable of association with the xcex22 subunit, but having a unique molecular weight and restricted in expression to a subset of differentiated tissue macrophages.
Antibodies generated by immunization of hamsters with murine dendritic cells resulted in two anti-integrin antibodies [Metlay, et al., J.Exp.Med. 171:1753-1771 (1990)]. One antibody, 2E6, immunoprecipitated a predominant heterodimer with subunits having approximate molecular weights of 180 kD and 90 kD in addition to minor bands in the molecular weight range of 150-160 kD. The second antibody, N418, precipitated another apparent heterodimer with subunits having approximate molecular weights of 150 kD and 90 Kd. Based on cellular adhesion blocking studies, it was hypothesized that antibody 2E6 recognized a murine counterpart to human CD18. While the molecular weight of the N418 antigen suggested recognition of a murine homolog to human CD11c/CD18, further analysis indicated that the murine antigen exhibited a tissue distribution pattern which was inconsistent with that observed for human CD11c/CD18.
The antigens recognized by the canine Ca11.8H2 antibody and the murine N418 antibody could represent a variant species (e.g., a glycosylation or splice variant) of a previously identified canine or murine xcex1 subunit. Alternatively, these antigens may represent unique canine and murine integrin xcex1 subunits. In the absence of specific information regarding primary structure, these alternatives cannot be distinguished.
In humans, CD11a/CD18 is expressed on all leukocytes. CD11b/CD18 and CD11c/CD18 are essentially restricted to expression on monocytes, granulocytes, macrophages and natural killer (NK) cells, but CD11c/CD18 is also detected on some B-cell types. In general, CD11a/CD18 predominates on lymphocytes, CD11b/CD18 on granulocytes and CD11c/CD18 on macrophages [see review, Arnaout, Blood 75:1037-1050 (1990)]. Expression of the a chains, however, is variable with regard to the state of activation and differentiation of the individual cell types [See review, Larson and Springer, Immunol.Rev. 114:181-217 (1990).]
The involvement of the xcex22 integrins in human immune and inflammatory responses has been demonstrated using monoclonal antibodies which are capable of blocking xcex22 integrin-associated cell adhesion. For example, CD11a/CD18, CD11b/CD18 and CD11c/CD18 actively participate in natural killer (NK) cell binding to lymphoma and adenocarcinoma cells [Patarroyo, et al., Immunol.Rev. 114:67-108 (1990)], granulocyte accumulation [Nourshargh, et al., J.Immunol. 142:3193-3198 (1989)], granulocyte-independent plasma leakage [Arfors, et al., Blood 69:338-340 (1987)], chemotactic response of stimulated leukocytes [Arfors, et al., supra] and leukocyte adhesion to vascular endothelium [Price, et al., J.Immunol. 139:4174-4177 (1987) and Smith, et al., J.Clin.Invest. 83:2008-2017 (1989)]. The fundamental role of xcex22 integrins in immune and inflammatory responses is made apparent in the clinical syndrome referred to as leukocyte adhesion deficiency (LAD), wherein clinical manifestations include recurrent and often life threatening bacterial infections. LAD results from heterogeneous mutations in the xcex22 subunit [Kishimoto, et al., Cell 50:193-202 (1987)] and the severity of the disease state is proportional to the degree of the deficiency in xcex22 subunit expression. Formation of the complete integrin heterodimer is impaired by the xcex22 mutation [Kishimoto, et al., supra]. Interestingly, at least one antibody specific for CD18 has been shown to inhibit human immunodeficiency virus type-1 (HIV-1) syncytia formation in vitro, albeit the exact mechanism of this inhibition is unclear [Hildreth and Orentas, Science 244:1075-1078 (1989)]. This observation is consistent with the discovery that a principal counterreceptor of CD11a/CD18, ICAM-1, is also a surface receptor for the major group of rhinovirus serotypes [Greve, et al., Cell 56:839 (1989)].
The significance of xcex22 integrin binding activity in human immune and inflammatory responses underscores the necessity to develop a more complete understanding of this class of surface proteins. Identification of yet unknown members of this subfamily, as well as their counterreceptors, and the generation of monoclonal antibodies or other soluble factors which can alter biological activity of the xcex22 integrins will provide practical means for therapeutic intervention in xcex22 integrin-related immune and inflammatory responses.
In one aspect, the present invention provides novel purified and isolated polynucleotides (e.g., DNA and RNA transcripts, both sense and anti-sense strands) encoding a novel human xcex22 integrin a subunit, xcex1d, and variants thereof (i.e., deletion, addition or substitution analogs) which possess binding and/or immunological properties inherent to xcex1d. Preferred DNA molecules of the invention include cDNA, genomic DNA and wholly or partially chemically synthesized DNA molecules. A presently preferred polynucleotide is the DNA as set forth in SEQ ID NO: 1, encoding the polypeptide of SEQ ID NO: 2. Also provided are recombinant plasmid and viral DNA constructions (expression constructs) which include xcex1d encoding sequences, wherein the xcex1d encoding sequence is operatively linked to a homologous or heterologous transcriptional regulatory element or elements.
Also provided by the present invention are isolated and purified mouse and rat polynucleotides which exhibit homology to polynucleotides encoding human xcex1d. A preferred mouse polynucleotide is set forth in SEQ ID NO: 52; a preferred rat polynucleotide is set forth in SEQ ID NO: 54.
As another aspect of the invention, prokaryotic or eukaryotic host cells transformed or transfected with DNA sequences of the invention are provided which express xcex1d polypeptide or variants thereof. Host cells of the invention are particularly useful for large scale production of xcex1d polypeptide, which can be isolated from either the host cell itself or from the medium in which the host cell is grown. Host cells which express xcex1d polypeptide on their extracellular membrane surface are also useful as immunogens in the production of xcex1d-specific antibodies. Preferably, host cells transfected with xcex1d will be co-transfected to express a xcex22 integrin subunit in order to allow surface expression of the heterodimer.
Also provided by the present invention are purified and isolated xcex1d polypeptides, fragments and variants thereof. Preferred xcex1d polypeptides are as set forth in SEQ ID NO: 2. Novel xcex1d products of the invention may be obtained as isolates from natural sources, but, along with xcex1d variant products, are preferably produced by recombinant procedures involving host cells of the invention. Completely glycosylated, partially glycosylated and wholly de-glycosylated forms of the xcex1d polypeptide may be generated by varying the host cell selected for recombinant production and/or post-isolation processing. Variant xcex1d polypeptides of the invention may comprise water soluble and insoluble xcex1d polypeptides including analogs wherein one or more of the amino acids are deleted or replaced: (1) without loss, and preferably with enhancement, of one or more biological activities or immunological characteristics specific for xcex1d; or (2) with specific disablement of a particular ligand/receptor binding or signalling function. Fusion polypeptides are also provided, wherein xcex1d amino acid sequences are expressed contiguously with amino acid sequences from other polypeptides. Such fusion polypeptides may possess modified biological, biochemical, and/or immunological properties in comparison to wild-type xcex1d. Analog polypeptides including additional amino acid (e.g., lysine or cysteine) residues that facilitate multimer formation are contemplated.
Also comprehended by the present invention are polypeptides and other non-peptide molecules which specifically bind to xcex1d. Preferred binding molecules include antibodies (e.g., monoclonal and polyclonal antibodies), counterreceptors (e.g., membrane-associated and soluble forms) and other ligands (e.g., naturally occurring or synthetic molecules), including those which competitively bind xcex1d in the presence of xcex1d monoclonal antibodies and/or specific counterreceptors. Binding molecules are useful for purification of xcex1d polypeptides and identifying cell types which express xcex1d. Binding molecules are also useful for modulating (i.e., inhibiting, blocking or stimulating) of in vivo binding and/or signal transduction activities of xcex1d.
Assays to identify xcex1d binding molecules are also provided, including in vitro assays such as immobilized ligand binding assays, solution binding assays, and scintillation proximity assays, as well as cell based assays such as di-hybrid screening assays, split hybrid screening assays, and the like. Cell based assays provide for a phenotypic change in a host cell as a result of specific binding interaction or disruption of a specific binding interaction, thereby permitting indirect quantitation or measurement of some specific binding interaction.
In vitro assays for identifying antibodies or other compounds that modulate the activity of xcex1d may involve, for example, immobilizing xcex1d or a natural ligand to which xcex1d binds, detectably labelling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the presence of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of xcex1d binding.
Another type of in vitro assay for identifying compounds that modulate the interaction between xcex1d and a ligand involves immobilizing xcex1d or a fragment thereof on a solid support coated (or impregnated with) a fluorescent agent, labeling the ligand with a compound capable of exciting the fluorescent agent, contacting the immobilized xcex1d with the labeled ligand in the presence and absence of a putative modulator compound, detecting light emission by the fluorescent agent, and identifying modulating compounds as those compounds that affect the emission of light by the fluorescent agent in comparison to the emission of light by the fluorescent agent in the absence of a modulating compound. Alternatively, the xcex1d ligand may be immobilized and xcex1d may be labeled in the assay.
A cell based assay method contemplated by the invention for identifying compounds that modulate the interaction between xcex1d and a ligand involves transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain, expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of xcex1d and either the DNA binding domain or the activating domain of the transcription factor, expressing in the host cells a second hybrid DNA sequence encoding part or all of the ligand and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion, evaluating the effect of a putative modulating compound on the interaction between xcex1d and the ligand by detecting binding of the ligand to xcex1d in a particular host cell by measuring the production of reporter gene product in the host cell in the presence or absence of the putative modulator, and identifying modulating compounds as those compounds altering production of the reported gene product in comparison to production of the reporter gene product in the absence of the modulating compound. Presently preferred for use in the assay are the lexA promoter, the lexA DNA binding domain, the GAL4 transactivation domain, the lacZ reporter gene, and a yeast host cell.
A modified version of the foregoing assay may be used in isolating a polynucleotide encoding a protein that binds to xcex1d by transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain, expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of xcex1d and either the DNA binding domain or the activating domain of the transcription factor, expressing in the host cells a library of second hybrid DNA sequences encoding second fusions of part or all of putative xcex1d binding proteins and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion, detecting binding of an xcex1d binding protein to xcex1d in a particular host cell by detecting the production of reporter gene product in the host cell, and isolating second hybrid DNA sequences encoding xcex1d binding protein from the particular host cell.
In a preferred embodiment utilizing the split hybrid assay, the invention provides a method to identify an inhibitor of binding between an xcex1d protein or fragment thereof and an xcex1d binding protein or binding fragment thereof comprising the steps of: (a) transforming or transfecting a host cell with a first DNA expression construct comprising a first selectable marker gene encoding a first selectable marker protein and a repressor gene encoding a repressor protein, said repressor gene under transcriptional control of a promoter; (b) transforming or transfecting said host cell with a second DNA expression construct comprising a second selectable marker gene encoding a second selectable marker protein and a third selectable marker gene encoding a third selectable marker protein, said third selectable marker gene under transcriptional control of an operator, said operator specifically acted upon by said repressor protein such that interaction of said repressor protein with said operator decreases expression of said third selectable marker protein; (c) transforming or transfecting said host cell with a third DNA expression construct comprising a fourth selectable marker gene encoding a fourth selectable marker protein and an xcex1d fusion protein gene encoding an xcex1d protein or fragment thereof in frame with either a DNA binding domain of a transcriptional activation protein or a transactivating domain of said transcriptional activation protein; (d) transforming or transfecting said host cell with a fourth DNA expression construct comprising a fifth selectable marker gene encoding a fifth selectable marker protein and a second fusion protein gene encoding an xcex1d binding protein or binding fragment thereof in frame with either the DNA binding domain of said transcriptional activation protein or the transactivating domain of said transcriptional activation protein, whichever is not included in first fusion protein gene; (e) growing said host cell under conditions which permit expression of said xcex1d protein or fragment thereof and said xcex1d binding protein or fragment thereof such that said xcex1d protein or fragment thereof and xcex1d binding protein or binding fragment thereof interact bringing into proximity said DNA binding domain and said transactivating domain reconstituting said transcriptional activating protein; said transcriptional activating protein acting on said promoter to increase expression of said repressor protein; said repressor protein interacting with said operator such that said third selectable marker protein is not expressed; (f) detecting absence of expression of said selectable gene; (g) growing said host cell in the presence of a test inhibitor of binding between said xcex1d protein or fragment thereof and said xcex1d binding protein or fragment thereof; and (h) comparing expression of said selectable marker protein in the presence and absence of said test inhibitor wherein decreased expression of said selectable marker protein is indicative of an ability of the test inhibitor to inhibit binding between said xcex1d protein or fragment thereof and said xcex1d binding protein or binding fragment thereof such that said transcriptional activating protein is not reconstituted, expression of said repressor protein is not increased, and said operator increases expression of said selectable marker protein.
The invention comprehends host cells wherein the various genes and regulatory sequences are encoded on a single DNA molecule as well as host cells wherein one or more of the repressor gene, the selectable marker gene, the xcex1d fusion protein gene, and the xcex1d binding protein gene are encoded on distinct DNA expression constructs. In a preferred embodiment, the host cells are transformed or transfected with DNA encoding the repressor gene, the selectable marker gene, the xcex1d fusion protein gene, and the d fusion binding protein gene, each encoded on a distinct expression construct. Regardless of the number of DNA expression constructs introduced, each transformed or transfected DNA expression construct further comprises a selectable marker gene sequence, the expression of which is used to confirm that transfection or transformation was, in fact, accomplished. Selectable marker genes encoded on individually transformed or transfected DNA expression constructs are distinguishable from the selectable marker under transcriptional regulation of the tet operator in that expression of the selectable marker gene regulated by the tet operator is central to the preferred embodiment; i.e., regulated expression of the selectable marker gene by the tet operator provides a measurable phenotypic change in the host cell that is used to identify a binding protein inhibitor. Selectable marker genes encoded on individually transformed or transfected DNA expression constructs are provided as determinants of successful transfection or transformation of the individual DNA expression constructs. Preferred host cells of the invention include transformed S. cerevisiae strains designated YI596 and YI584 which were deposited Aug. 13, 1996 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, and assigned Accession Numbers ATCC 74384 and ATCC 74385, respectively.
The host cells of the invention include any cell type capable of expressing the xcex1d and xcex1d binding proteins required as described above and which are capable of being transformed or transfected with functional promoter and operator sequences which regulate expression of the heterologous proteins also as described. In a preferred embodiment, the host cells are of either mammal, insect or yeast origin. Presently, the most preferred host cell is a yeast cell. The preferred yeast cells of the invention can be selected from various strains, including the S. cerevisiae yeast transfonnants described in Table 1. Alternative yeast specimens include S.pombe, K.lactis, P.pastoris, S.carlsbergensis and C.albicans. Preferred mammalian host cells of the invention include Chinese hamster ovary (CHO), COS, HeLa, 3T3, CV1, LTK, 293T3, Rat1, PC12 or any other transfectable cell line of human or rodent origin. Preferred insect cell lines include SF9 cells.
In a preferred embodiment, the selectable marker gene is regulated by an operator and encodes an enzyme in a pathway for synthesis of a nutritional requirement for said host cell such that expression of said selectable marker protein is required for growth of said host cell on media lacking said nutritional requirement. Thus, as in a preferred embodiment where a repressor protein interacts with the operator, transcription of the selectable marker gene is down-regulated and the host cells are identified by an inability to grow on media lacking the nutritional requirement and an ability to grow on media containing the nutritional requirement. In a most preferred embodiment, the selectable marker gene encodes the HIS3 protein, and host cells transformed or transfected with a HIS3-encoding DNA expression construct are selected following growth on media in the presence and absence of histidine. The invention, however, comprehends any of a number of alternative selectable marker genes regulated by an operator. Gene alternatives include, for example, URA3, LEU2, LYS2 or those encoding any of the multitude of enzymes required in various pathways for production of a nutritional requirement which can be definitively excluded from the media of growth. In addition, conventional reporter genes such as chloramphenicol acetyltransferase (CAT), firefly luciferase, xcex2-galactosidase (xcex2-gal), secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), human growth hormone (hGH), xcex2-glucuronidase, neomycin, hygromycin, thymidine kinase (TK) and the like may be utilized in the invention.
In the preferred embodiment, the host cells include a repressor protein gene encoding the tetracycline resistance protein which acts on the tet operator to decrease expression of the selectable marker gene. The invention, however, also encompasses alternatives to the tet repressor and operator, for example, E.coli trp repressor and operator, his repressor and operator, and lac operon repressor and operator.
The DNA binding domain and transactivating domain components of the fusion proteins may be derived from the same transcription factor or from different transcription factors as long as bringing the two domains into proximity through binding between xcex1d and the xcex1d binding protein permits formation of a functional transcriptional activating protein that increases expression of the repressor protein with high efficiency. A high efficiency transcriptional activating protein is defined as having both a DNA binding domain exhibiting high affinity binding for the recognized promoter sequence and a transactivating domain having high affinity binding for transcriptional machinery proteins required to express repressor gene mRNA. The DNA binding domain component of a fusion protein of the invention can be derived from any of a number of different proteins including, for example, LexA or Gal4. Similarly, the transactivating component of the invention""s fusion proteins can be derived from a number of different transcriptional activating proteins, including for example, Gal4 or VP16.
The promoter sequence of the invention which regulates transcription of the repressor protein can be any sequence capable of driving transcription in the chosen host cell. The promoter may be a DNA sequence specifically recognized by the chosen DNA binding domain of the invention, or any other DNA sequence with which the DNA binding domain of the fusion protein is capable of high affinity interaction. In a preferred embodiment of the invention, the promoter sequence of the invention is either a HIS3 or alcohol dehydrogenase (ADH) promoter. In a presently most preferred embodiment, the ADH promotor is employed in the invention. The invention, however, encompasses numerous alternative promoters, including, for example, those derived from genes encoding HIS3, ADH, URA3, LEU2 and the like.
The methods of the invention encompass any and all of the variations in host cells as described above. In particular, the invention encompasses a method wherein: the host cell is a yeast cell; the selectable marker gene encodes HIS3; transcription of the selectable marker gene is regulated by the tet operator; the repressor protein gene encodes the tetracycline resistance protein; transcription of the tetracycline resistance protein is regulated by the HIS3 promoter; the DNA binding domain is derived from LexA; and the transactivating domain is derived from VP16. In another embodiment, the invention encompasses a method wherein: the host cell is a yeast cell; the selectable marker gene encodes HIS3; transcription of the selectable marker gene is regulated by the tet operator; the repressor protein gene encodes the tetracycline resistance protein; transcription of the tetracycline resistance protein is regulated by the alcohol dehydrogenase promoter; the DNA binding domain is derived from LexA; and the transactivating domain is derived from VP16.
In alternative embodiments of the invention wherein the host cell is a mammalian cell, variations include the use of mammalian DNA expression constructs to encode the xcex1d and xcex1d binding fusion genes, the repressor gene, and the selectable marker gene, and use of selectable marker genes encoding antibiotic or drug resistance markers (i.e., neomycin, hygromycin, thymidine kinase).
There are at least three different types of libraries used for the identification of small molecule modulators. These include: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
Chemical libraries consist of structural analogs of known compounds or compounds that are identified as xe2x80x9chitsxe2x80x9d via natural product screening. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
Hybridoma cell lines which produce antibodies specific for xcex1d are also comprehended by the invention. Techniques for producing hybridomas which secrete monoclonal antibodies are well known in the art. Hybridoma cell lines may be generated after immunizing an animal with purified xcex1d, variants of xcex1d or cells which express xcex1d or a variant thereof on the extracellular membrane surface. Immunogen cell types include cells which express xcex1d in vivo, or transfected prokaryotic or eukaryotic cell lines which normally do not normally express xcex1d in vivo. Presently preferred antibodies of the invention are secreted by hybridomas designated 169A, 169B, 170D, 170F, 170E, 170X, 170H, 188A, 188B, 188C, 188E, 188F, 188G, 188I, 188J, 188K, 188L, 188M, 188N, 188P, 188R, 188T, 195A, 195C, 195D, 195E, 195H, 197A-1, 197A-2, 197A-3, 197A-4, 199A, 199H, 199M, 205A, 205C, 205E, 212A, 212D, 217G, 217H, 217I, 217K, 217L, 217M, 226A, 226B, 226C, 226D, 226E, 226F, 226G, 226H, 2261, 236A, 236B, 236C, 236F, 236G, 236H, 236I, 236K, 237L, 236M, 240F, 240G, 240H, and 236L.
The value of the information contributed through the disclosure of the DNA and amino acid sequences of xcex1d is manifest. In one series of examples, the disclosed xcex1d CDNA sequence makes possible the isolation of the human xcex1d genomic DNA sequence, including transcriptional control elements for the genomic sequence. Identification of xcex1d allelic variants and heterologous species (e.g., rat or mouse) DNAs is also comprehended. Isolation of the human xcex1d genomic DNA and heterologous species DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of the xcex1d cDNA sequence as a probe to screen an appropriate library. Alternatively, polymerase chain reaction (PCR) using oligonucleotide primers that are designed based on the known cDNA sequence can be used to amplify and identify genomic xcex1d DNA sequences. Synthetic DNAs encoding the xcex1d polypeptide, including fragments and other variants thereof, may be produced by conventional synthesis methods.
DNA sequence information of the invention also makes possible the development, by homologous recombination or xe2x80x9cknockoutxe2x80x9d strategies [see, e.g., Kapecchi, Science 244:1288-1292 (1989)], to produce rodents that fail to express a functional xcex1d polypeptide or that express a variant xcex1d polypeptide. Such rodents are useful as models for studying the activities of xcex1d and xcex1d modulators in vivo.
DNA and amino acid sequences of the invention also make possible the analysis of xcex1d epitopes which actively participate in counterreceptor binding as well as epitopes which may regulate, rather than actively participate in, binding. Identification of epitopes which may participate in transmembrane signal transduction is also comprehended by the invention.
DNA of the invention is also useful for the detection of cell types which express xcex1d polypeptide. Standard DNA/RNA hybridization techniques which utilize xcex1d DNA to detect xcex1d RNA may be used to determine the constitutive level of xcex1d transcription within a cell, as well as changes in the level of transcription in response to internal or external agents. Identification of agents which modify transcription and/or translation of xcex1d can, in turn, be assessed for potential therapeutic or prophylactic value. DNA of the invention also makes possible in situ hybridization of xcex1d DNA to cellular RNA to determine the cellular localization of xcex1d specific messages within complex cell populations and tissues.
DNA of the invention is also useful for identification of non-human polynucleotide sequences which display homology to human xcex1d sequences. Possession of non-human xcex1d DNA sequences permits development of animal models (including, for example, transgenic models) of the human system.
As another aspect of the invention, monoclonal or polyclonal antibodies specific for xcex1d may be employed in immunohistochemical analysis to localize xcex1d to subcellular compartments or individual cells within tissues. Immunohistochemical analyses of this type are particularly useful when used in combination with in situ hybridization to localize both xcex1d mRNA and polypeptide products of the xcex1d gene.
Identification of cell types which express xcex1d may have significant ramifications for development of therapeutic and prophylactic agents. It is anticipated that the products of the invention related to xcex1d can be employed in the treatment of diseases wherein macrophages are an essential element of the disease process. Animal models for many pathological conditions associated with macrophage activity have been described in the art. For example, in mice, macrophage recruitment to sites of both chronic and acute inflammation is reported by Jutila, et al., J.Leukocyte Biol. 54:30-39 (1993). In rats, Adams, et al., [Transplantation 53:1115-1119(1992) and Transplantation 56:794-799 (1993)] describe a model for graft arteriosclerosis following heterotropic abdominal cardiac allograft transplantation. Rosenfeld, et al., [Arteriosclerosis 7:9-23 (1987) and Arteriosclerosis 7:24-34 (1987)] describe induced atherosclerosis in rabbits fed a cholesterol supplemented diet. Hanenberg, et al., [Diabetologia 32:126-134 (1989)] report the spontaneous development of insulin-dependent diabetes in BB rats. Yamada et al., [Gastroenterology 104:759-771 (1993)] describe an induced inflammatory bowel disease, chronic granulomatous colitis, in rats following injections of streptococcal peptidoglycan-polysaccharide polymers. Cromartie, et al., [J.Exp.Med. 146:1585-1602 (1977)] and Schwab, et al., [Infection and Immunity 59:4436-4442 (1991)] report that injection of streptococcal cell wall protein into rats results in an arthritic condition characterized by inflammation of peripheral joints and subsequent joint destruction. Finally, Huitinga, et al., [Eur.J.Immunol 23:709-715 (1993) describe experimental allergic encephalomyelitis, a model for multiple sclerosis, in Lewis rats. In each of these models, xcex1d antibodies, other xcex1d binding proteins, or soluble forms of xcex1d are utilized to attenuate the disease state, presumably through inactivation of macrophage activity.
Pharmaceutical compositions for treatment of these and other disease states are provided by the invention. Pharmaceutical compositions are designed for the purpose of inhibiting interaction between xcex1d and its ligand(s) and include various soluble and membrane-associated forms of xcex1d (comprising the entire xcex1d polypeptide, or fragments thereof which actively participate in xcex1d binding), soluble and membrane-associated forms of xcex1d binding proteins (including antibodies, ligands, and the like), intracellular or extracellular modulators of xcex1d binding activity, and/or modulators of xcex1d and/or xcex1d-ligand polypeptide expression, including modulators of transcription, translation, post-translational processing and/or intracellular transport.
The invention also comprehends methods for treatment of disease states in which xcex1d binding, or localized accumulation of cells which express xcex1d, is implicated, wherein a patient suffering from said disease state is provided an amount of a pharmaceutical composition of the invention sufficient to modulate levels of xcex1d binding or to modulate accumulation of cell types which express xcex1d. The method of treatment of the invention is applicable to disease states such as, but not limited to, Type I diabetes, atherosclerosis, multiple sclerosis, asthma, psoriasis, lung inflammation, acute respiratory distress syndrome and rheumatoid arthritis.
The invention also provides methods for inhibiting macrophage infiltration at the site of a central nervous system injury comprising the step of administering to an individual an effective amount of an anti-xcex1d monoclonal antibody. In one aspect, the methods comprise use of an anti-xcex1d monoclonal antibody that blocks binding between xcex1d and a binding partner. In one embodiment, the binding partner is VCAM-1. In a preferred embodiment, the anti-xcex1d monoclonal antibody is selected from the group consisting of the monoclonal antibody secreted by hybridoma 226H and the monoclonal antibody secreted by hybridoma 236L. In a most preferred embodiment, methods of the invention are for a central nervous system injury which is a spinal cord injury.
The invention further provides methods for reducing inflammation at the site of a central nervous system injury comprising the step of administering to an individual an effective amount of an anti-xcex1d monoclonal antibody. In one aspect, the methods comprise use of an anti-xcex1d monoclonal antibody that blocks binding between xcex1d and a binding partner. In one embodiment, the binding partner is VCAM-1. In a preferred embodiment, the anti-xcex1d monoclonal antibody is selected from the group consisting of the monoclonal antibody secreted by hybridoma 226H and the monoclonal antibody secreted by hybridoma 236L. In a most preferred embodiment, methods of the invention are for a central nervous system injury which is a spinal cord injury.
Hybridomas 226H and 236L were received on Nov. 11, 1998 by the American Type Culture Collection, 10801 University Boulevard, Masassas, Va. 20110-2209 under terms of the Budapest Treaty and assigned Accession Nos: HB-12592 and HB-12593, respectively.
The invention also provides methods for modulating TNFxcex1 release from macrophage or splenic phagocytes comprising the step of contacting said phagocytes with an affective amount of an immunospecific xcex1d monoclonal antibody. In a preferred aspect, the method methods of the invention comprise an anti-monoclonal antibody that inhibits TNFxcex1 release. In a preferred embodiment, the methods of the invention comprise use of an immunospecific anti-xcex1d monoclonal antibody that is selected from the group consisting of the monoclonal antibody secreted by hybridoma 205C and the monoclonal antibody secreted by hybridoma 205E.
Methods of the invention are contemplated wherein useful antibodies include fragments of anti-xcex1d monoclonal antibodies, including for example, Fab or F(abxe2x80x2)2 fragments. Methods utilizing modified antibodies are also embraced by the invention. Modified antibodies include, for example, single chain antibodies, chimeric antibodies, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention, as well as humanized antibodies. Methods comprising use of human antibodies are also contemplated. Techniques for identifying and isolating human antibodies are disclosed infra.
The invention also provides methods for inhibiting macrophage infiltration at the site of a central nervous system injury comprising the step of administering to an individual an effective amount of a small molecule that inhibits xcex1d binding. In particular, the methods of the invention comprising a central nervous system injury which is a spinal cord injury. Small molecules specific for xcex1d binding are identified and isolated from libraries as discussed above.
The invention further provides methods for reducing inflammation at the site of a central nervous system injury comprising the step of administering to an individual an effective amount of a small molecule that inhibits xcex1d binding. In particular, the methods of the invention comprising a central nervous system injury which is a spinal cord injury. Small molecules specific for xcex1d binding are identified and isolated from libraries as discussed above.
The invention further embraces methods to detect and diagnose Crohn""s disease comprising the steps of obtaining tissue samples from a patient; staining the sample with anti-xcex1d monoclonal antibodies, and comparing the staining pattern to that on tissue obtained from a known normal donor. In instances wherein staining differences between the two tissue samples can be detected, the patient can be further tested for possible Crohn""s disease.
The invention also contemplates use of xcex1d as a target for removal of pathogenic cell populations expressing xcex1d on the cell surface. In one aspect, the hypervariable region of an xcex1d monoclonal antibody is cloned and expressed in the context of a complement-fixing human isotype. Cloning the hypervariable region in this manner will provide a binding partner for xcex1d, which unpon binding in vivo, will lead to complement binding and subsequent cell death. Alternatively, the anti-xcex1d monoclonal antibody is conjugated to a cytotoxic compound and binding of the antibody to xcex1d on the pathogenic cell type leads to cell death.
The invention also provide methods for promoting locomoter recovery or inhibiting locomoter damage or limiting locomoter impairment following spinal cord injury by administering an effective amount of an anti-xcex1d monoclonal antibody to a spinal cord injury victim. Also contemplated are methods of limiting autonomic and sensory dysfunction following spinal cord injury by administration of an effective amount of an anti-xcex1d monoclonal antibody to a spinal cord injury victim. In one embodiment, the anti-xcex1d monoclonal antibody is 217L or 226H antibody. In another embodiment the anti-xcex1d monoclonal antibody competes with 217L or 226H for binding to xcex1d. In a further embodiment, the anti-xcex1d monoclonal antibody inhibits xcex1d binding to an xcex1d ligand. Contemplated xcex1d ligands include ICAM-R and VCAM-1. Exemplary spinal cord injuries treated by these methods include those involving compression to the spinal cord.